Post-Column Derivatizations of Amino Acids

Even today, the post-coltunn derivatization with ninhydrin introduced by Spademan, Stein, and Moore [24] represents the most common detechon method for quantitative amino acid analysis. As a strong oxidant, ninhydrin reacts with the a-amino groups of eluting amino acids at temperatures around 130°C, according to Eq. (125), releasing ammonia and carbon dioxide. 

An aldehyde shorter by one C-atom and hydrindantin, the reduced form of ninhydrin, are formed in this reaction. Hydrintantin reacts with ammonia and a second molecule of ninhydrin to form a red dye called Ruhemann s purple. This dye has an absorption maximum at 570 nm (see Fig. 5-28). 

Secondary amino acids, so-called imino adds such as proline and hydroxy-proline, do not possess an a-amino group, and react with ninhydrin to form a yellow product which is usually detected at 440 nm. Therefore, amino add analyzers are equipped with a photometric detedor capable of measuring at two dif ferent wavelengths (570 nm and 440 nm). The sensitivity of this detection method is about 200 pmol. 

The subsequent fluorescence detection is carried out at an excitation wavelength of 340 nm the emission is measured at 455 nm. The addition of thiols such as 2-mercaptoethanol increases the fluorescence yield. However, OPA only reacts with primary amino acids. Secondary amino acids can only be detected after their oxidation (for example with hypochlorite or chloramine T) [45]. In practical applications, this creates significant difficulties. 

An interesting derivatization method was recently described by Jenke and Brown [46]. They mixed the colunm effluent with a buffered solution of 5,5 -dithiobis(2-nitrobenzoic acid) (DTNB), yielding a strongly yellow-colored chromophore, which can be detected photometrically at 412 nm  

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The post-column derivatization of amino acids by the ninhydrin technique is a well known method for routine analysis of amino acids [7-9]. The amino acids are usually separated by ion-exchange chromatography and then converted into UV-absorbing derivatives for quantitation. The ninhydrin reaction is often used for TLC detection of amino acids and proteins. 

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